Canola is the seed derived from any of the Brassica species B. napus, B. campestris/rapa, and certain varieties of B. juncea. Canola oil is high in monounsaturated fats, moderate in polyunsaturated fats, and low in saturated fats, having the lowest level of saturated fat of any vegetable oil. Thus canola oil is an important dietary option for lowering serum cholesterol in humans. In addition, the protein meal which is the byproduct of canola oil production has a high nutritional content and is used in animal feeds.
Imidazolinone and sulfonylurea herbicides are widely used in modern agriculture due to their effectiveness at very low application rates and relative non-toxicity in animals. Both of these herbicides act by inhibiting acetohydroxyacid synthase (AHAS; EC 4.1.3.18, also known as acetolactate synthase or ALS), the first enzyme in the synthetic pathway of the branched chain amino acids valine, leucine and isoleucine. Several examples of commercially available imidazolinone herbicides are PURSUIT® (imazethapyr), SCEPTER® (imazaquin) and ARSENAL® (imazapyr). Examples of sulfonylurea herbicides are chlorsulfuron, metsulfuron methyl, sulfometuron methyl, chlorimuron ethyl, thifensulfuron methyl, tribenuron methyl, bensulfuron methyl, nicosulfuron, ethametsulfuron methyl, rimsulfuron, triflusulfuron methyl, triasulfuron, primisulfuron methyl, cinosulfuron, amidosulfuron, fluzasulfuron, imazosulfuron, pyrazosulfuron ethyl and halosulfuron.
Due to their high effectiveness and low toxicity, imidazolinone herbicides are favored for application to many crops, including canola, by spraying over the top of a wide area of vegetation. The ability to spray an herbicide over the top of a wide range of vegetation decreases the costs associated with plantation establishment and maintenance and decreases the need for site preparation prior to use of such chemicals. Spraying over the top of a desired tolerant species also results in the ability to achieve maximum yield potential of the desired species due to the absence of competitive species. However, the ability to use such spray-over techniques is dependent upon the presence of imidazolinone resistant species of the desired vegetation in the spray over area. In addition, because residual imidazolinones persist in a sprayed field, a variety of resistant species is advantageous for crop rotation purposes.
Unfortunately, the Brassica species which are the source of canola are closely related to a number of broad leaf cruciferous weeds, for example, stinkweed, ball mustard, wormseed mustard, hare's ear mustard, shepherd's purse, common peppergrass, flixweed, and the like. Thus it was necessary to develop Brassica cultivars which are tolerant or resistant to the imidazolinone herbicides. Swanson, et al. (1989) Theor. Appl. Genet. 78, 525-530 discloses B. napus mutants P1 and P2, developed by mutagenesis of microspores of B. napus (cv ‘Topas’), which demonstrated tolerance to the imidazolinone herbicides PURSUIT® and ASSERT® at levels approaching ten times the field-recommended rates. The homozygous P2 mutant produced an AHAS enzyme which was 500 times more tolerant to PURSUIT® than wild type enzyme, while the AHAS enzyme from the homozygous P1 mutant was only slightly more tolerant than the wild type enzyme. In field trials, the P1, P2, and P1×P2 hybrid withstood ASSERT® applications up to 800 g/ha with no loss of yield. The P1 and P2 mutations were unlinked and semidominant, and P1×P2 crosses tolerated levels of PURSUIT® higher than those tolerated by either homozygous mutant. Imidazolinone-tolerant cultivars of B. napus were developed from the P1×P2 mutants and have been sold as CLEARFIELD® canola. See also, Canadian patent application number 2,340,282; Canadian patent number 1,335,412, and European patent number 284419.
Rutledge, et al. (1991) Mol. Gen. Genet. 229, 31-40) discloses the nucleic acid sequence of three of the five genes encoding AHAS isoenzymes in B. napus, AHAS1, AHAS2, and AHAS3. Rutledge, et al. discusses the mutants of Swanson, et al. and predicts that the two alleles that conferred resistance to imidazolinone herbicides correspond to AHAS1 and AHAS3. Hattori et al. (1995) Mol. Gen. Genet. 246, 419-425 disclose a mutant allele of AHAS3 from a mutant B. napus cv Topas cell suspension culture line in which a single nucleotide change at codon 557 leading to an amino acid change from tryptophan to leucine confers resistance to sulfonylurea, imidazolinone, and triazolopyrimidine herbicides. Codon 557 of Hattori, et al. corresponds to codon 556 of the AHAS3 sequence disclosed in Rutledge, et al., supra, and to codon 556 of the AHAS3 sequence set forth as GENBANK accession number gi/17775/emb/Z11526/.
A single nucleotide mutation at codon 173 in a B. napus ALS gene, corresponding to AHAS2 of Rutledge et al, supra, leads to a change from Pro to Ser (Wiersma et al. (1989) Mol. Gen. Genet. 219, 413-420). The mutant B. napus AHAS2 gene was transformed into tobacco to produce a chlorsulfuron tolerant phenotype.
U.S. Pat. Nos. 6,114,116 and 6,358,686 disclose nucleic acid sequences from B. napus and B. oleracea containing polymorphisms, none of which appears to correspond to the polymorphism disclosed in Hattori, et al., supra.
For commercially relevant Brassica cultivars, it is necessary to ensure that each lot of herbicide-resistant seed contains all mutations necessary to confer herbicide tolerance. A method is needed to detect mutations in Brassica AHAS1 and AHAS3 genes that confer increased imidazolinone tolerance to commercial cultivars.